Significant advances have been made in clinical cancer immunotherapy. Nevertheless, precursor frequency of antigen-reactive T cells remains an important limiting factor in achieving higher clinical response rates. We propose a translational investigation in which a high affinity MART-1 T cell receptor (TCR) and the HSVsr39tk PET reporter/imaging gene are introduced into CDS T cells from metastatic melanoma patients using a lentiviral vector. This vector will be manufactured at the Indiana University National Gene Vector Laboratory. These TCR/tk-engineered T cells will be reintroduced to the patient after a lymphodepleting, but nonmyeloablative conditioning regimen. These adoptively transferred cells will be supported in vivo by systemic interleukin-2 and MART26-35 peptide pulsed dendritic cell vaccines. In this dose-escalation phase I clinical trial, already approved by the NIH Recombinant DNA Advisory Committee, safety and feasibility will be primary end-points, transgenic T cell persistence and in vivo PET imaging will be secondary end-points and clinical response the tertiary end-point. Two specific aims are proposed. In the first, the phase I trial will be conducted with safety, immunological and clinical response end-points. The second aim will focus on biological imaging: clinical trial imaging, quantitative animal modeling using engineered human T cells and assessment of sr39tk-specific immune responses. In this first-in-human clinical investigation, we will utilize a lentiviral vector encoding three transgenes (a, p MART TCR chains, HSV sr39tk) to noninvasively and serially image antitumor immune responses in man. This application is the byproduct of an ongoing collaboration between investigators from UCLA, Caltech, Children's Hospital of Los Angeles and USC. Although the trial will be conducted at UCLA, it takes advantage of the science and infrastructure of 3 research universities, 8 academic departments, 2 gene medicine programs, 3 cancer centers and 4 institutes (Molecular Imaging, Stem Cell, Molecular Medicine, AIDS). Lay summary. We propose to conduct a clinical gene therapy trial in patients with metastatic melanoma. We will use a crippled viral vector to introduce three genes into white blood cells isolated from the patients. Two of these genes will cause these white blood cells to produce a receptor on their surface that will recognize melanoma cells in patients, allowing them to be killed. The third gene will allow these white blood cells to be visualized in patients using a noninvasive PET scan. [unreadable] [unreadable] [unreadable]